Use of hydrogenotrophic acetogenic strains for preventing or treating digestive disorders

ABSTRACT

The invention concerns the use of non-pathogenic hydrogenotrophic acetogenic bacterial strains for preparing a composition for treating or preventing gastrointestinal disorders associated with productions of digestive gases and/or for modulating the microbial balance of the digestive ecosystem in a mammal. The invention also concerns said pharmaceutical or food compositions, and the methods for monitoring and preparing said strains.

The invention relates to the use of nonpathogenic, hydrogenotrophic,acetogenic bacterial strains for preparing a composition for treating orpreventing gastrointestinal disorders associated with digestive gasproduction, and/or for modulating the microbial balance of the digestiveecosystem in a mammal.

The prevalence of functional digestive disorders or functionalgastrointestinal disorders in the Western population is very high sinceit is estimated that they affect approximately 25% to 30% of the adultpopulation. In addition, these digestive disorders represent one of themain causes of consultation in gastroenterology (approximately 50% ofconsultations). The symptoms of these intestinal disorders are diverse,such as modification of intestinal transit, meteorism, abdominal painand bloating. The cause of these functional disorders for the momentremains poorly defined, but it is estimated that the gases producedduring digestion in the colon play an important role in generatingcertain symptoms such as flatulence excess, abdominal distension(bloating) and associated pain. Some treatments have been proposed, forinstance active charcoal, simethicone, smectite, antispasmodics and alsocertain food supplements based on ferments (Saccharomyces cerevisiae,Bifidobacterium, Lactobacillus), on plants or on fiber (oligofructose,fennel, algae, oats, citrus fruits, etc.), or having a mineral structure(octalite, etc.). These treatments are, however, poorly effective on thesymptoms linked to gas formation in the colon, and do not actselectively. The present invention proposes to remedy the drawbacks ofthe prior art, both in terms of treatment and in terms of preventingdigestive discomfort associated with production of gas in the colon.

To do this, the invention is based both on the physiologicalcharacteristics of hydrogenotrophic acetogenic bacteria, namely theirability to reduce the total volume of digestive fermentation gases (H₂and CO₂), and on their nutritional diversity which confers on them aconsiderable ecological advantage in the digestive ecosystem compared toother hydrogenotrophic microorganisms.

In humans, dietary carbohydrates which escape digestion and absorptionin the small intestine arrive in the colon where they are fermented by acomplex microflora. This anaerobic degradation of organic matterproduces terminal metabolites in the form of volatile fatty acids havingmetabolic (acetate, propionate) or trophic (butyrate) properties andalso gases (H₂, CO₂ and, in some individuals, CH₄).

Among these fermentation gases, H₂ plays an important role in themaintaining and the effectiveness of degradation of organic matter inthe human colon. Some H₂ is eliminated via the respiratory and rectalpathways, but most of this gas is reutilized in situ by the intestinalflora. The latter, called hydrogenotrophic flora, is composed ofacetogenic bacteria, of sulfur-reducing bacteria and of methanogenicarchaea.

Sulfur-reducing bacteria are found in the digestive microflora of allindividuals (Pochard et al. (1992) FEMS Microbiol. Lett. 98 p 225). Theysynthesize H₂S, which is a potentially toxic product for eukaryoticcells, and which is thought to be involved in some diseases of thedigestive system, in particular ulcerative colitis (Roediger et al.(1993), Gastroenterology, 104, p 802).

Methanogenic archaea produce CH₄, which is a nontoxic gas. This methaneproduction is only observed in a fraction of the human population(approximately 21% of adult Indians, 95% of the rural population ofadolescents in black Africa and 40% of the Western population) and it iseliminated via the respiratory pathway and in flatulence (Segal et al.(1988) Gut 29 p 608; Pochart et al. (1992) FEMS Microbiol. Lett. 98 p225). These individuals, called methane excreters, harbor a very largepopulation of methanogenic archae (>10⁸/g of dry fecal extract) (Durandet al. (1996) in: Mälkki Y and Cummings J H (eds Official Publicationsof the European Communities, p 58). In these individuals, methanogenesisis the main pathway for elimination of H₂.

Individuals who are not methane excreters re-use H₂ via alternativemechanisms, among which is reductive acetogenesis. This pathwayconstitutes a major metabolic process for using H₂ in non-methaneexcreters.

Studies have indeed shown that the fecal microflora ofnon-methane-excreting individuals mainly metabolizes H₂ and CO₂ toacetate, whereas that of methane-excreting individuals uses H₂ and CO₂to form methane (Lajoie et al. (1988) Appl. Environ. Microbiol. 54 p2733; Bernalier et al. (1996) FEMS Microbiol. Ecol. p 193). In parallel,Doré et al. (1995 FEMS Microbiol. Ecol. 17 p 279) have shown theexistence of a negative correlation between the number of methanogenicarchaea and that of acetogenic bacteria in the human colon.Non-methane-excreting individuals therefore harbor little or nomethanogenic archaea in the colon, which would allow maximum expressionof their acetogenic activity (Lajoie et al. (1988) Appl. Environ.Microbiol. 54 p 2733; Bernalier et al. (1996) FEMS Microbiol. Ecol. 19 p193).

The hydrogenotrophic, acetogenic flora is characterized by greattaxonomic diversity. It is composed of bacterial species belonging inparticular to the Clostridium, Ruminococcus and Streptococcus genera(Bernalier et al. (1996) Curr. Microbiol. 33 p 94) and also of certainspecies of the Eubacterium genus (Schink (1994) in: Drake H L (ed)Acetogenesis. New York: Chapman and Hall p 197).

The term “hydrogenotrophic, acetogenic bacteria” is intended to meanbacterial species which use the reductive pathway for acetate synthesis(or Wood-Ljungdahl pathway) to produce this metabolite when they growautotrophically using H₂/CO₂ and also when they grow heterotrophicallyusing an organic substrate. These hydrogenotrophic, acetogenic bacteriahave indeed a large nutritional capacity and, besides using H₂/CO₂, arecapable of fermenting a considerable number of saccharides and oforganic compounds (Bernalier et al (1996), Curr. Microbiol., 33 p 94).

The hydrogenotrophic, acetogenic bacterial strains according to theinvention produce short-chain fatty acids (SCFA), in particular acetate,from H₂ and CO₂ gases. This production of SCFAs has a physiologicaladvantage for the host, such as the prevention (protection) or treatmentof diverse pathologies (see below).

The simultaneous presence of organic compounds and of H₂/CO₂ in theculture medium (conditions equivalent to those encountered in the humancolon) may result in simultaneous use of the two substrates via thehydrogenotrophic, acetogenic strain (Breznak and Blum (1991), Arch.Microbiol., 156 p 105). This phenomenon, called mixotrophy, allows thebacterium to have a higher energetic yield and therefore to grow morerapidly.

This ability to consume H₂/CO₂ combined with the ability to use a largenumber of organic substrates and also the ability to grow by mixotrophytherefore confers a considerable ecological advantage on acetogenicbacteria compared to populations of methanogens which use only a limitednumber of substrates (H₂, formate), and sulfur-reducing populationswhich are dependent on the presence of sulfate for their H₂ metabolism.

It has been shown that the use of certain probiotic preparations,containing bacteria such as propionic bacteria, lactobacilli and/orbifidobacteria, makes it possible to modify the flora in the colon ofcertain patients (Bougle et al. (1999) Scand. J. Gastroenterol. 34 p144; Venturi et al. (1999) Aliment. Pharmacol. Ther. 13 p 1103).

The use of acetogenic bacteria as probiotics as defined by Fuller (1989,J. Appl. Bact., 66 p 365), in preparations which can be used as foodmedicaments or as food supplements, therefore proves to be aparticularly innovative pathway of interest, since their ability tometabolize H₂/CO₂ would make it possible to optimize fermentations inthe colon by decreasing the total volume of fermentation gases and byproducing acetate, a source of energy which can be metabolized by thehost. The reduction of the digestive gases would thus be an effectivemeans for preventing and/or treating digestive disorders associated withaccumulation of these gases.

An object of the present invention is therefore the use ofnonpathogenic, hydrogenotrophic, acetogenic strains, for regulating theabovementioned digestive disorders and/or modulating the balance of themicrobial flora in a mammal.

The mammals according to the present invention are preferablymonogastric mammals, as opposed to polygastric mammals such asruminants. Felines and canines are particularly intended, especiallydomestic mammals (cats and dogs), and also humans.

The term “nonpathogenic” is intended to mean a microbial species forwhich no pathology of the host associated with its presence has beendemonstrated (strain GRAS=Generally Recognized As Safe).

Such a use may be envisioned in various ways. The present inventionrelates to a prophylactic or therapeutic use, in order to prevent and/ortreat certain disorders of the digestive system. This prevention and/ortreatment may be carried out via regulation of the gases produced in thecolon, through modulating the microbial flora. A use of this type may beenvisioned under the direction of a physician or a health professional.In this case, the health professional decides upon the dose, theduration of treatment and also a possible combination of thenonpathogenic, hydrogenotrophic, acetogenic strain with other activeprinciples effective in preventing and/or treating the digestivedisorders targeted. Such a use may also require monitoring of thenonpathogenic, hydrogenotrophic, acetogenic strain using a method ofanalysis according to the invention, as defined later.

The present invention also relates to a therapeutic and/or prophylacticuse in which the user, himself or herself, decides upon theadministration of the nonpathogenic, hydrogenotrophic, acetogenicstrain. The desired aim is then to decrease the discomfort of the user,who wishes, for example, to improve his or her quality of life.

Specifically, the digestive disorders targeted by the present inventionaffect the quality of life of the patients who suffer therefrom. Thedegree of digestive discomfort engendered and/or the capacity of eachindividual to put up with these disorders determine(s) whether or notaffected individuals consult a physician.

In particular, the invention relates to the use of hydrogenotrophic,acetogenic strains, for preparing a composition for the followingapplications:

-   (1) preventing and/or treating digestive functional disorders,-   (2) modulating the balance of the microbial flora in the colon by    advantageously promoting the activity of the acetogenic bacterial    flora, in particular to the detriment of the methanogenic and    sulfur-reducing bacterial flora.

The latter point has the advantage:

-   (1) of decreasing the formation of CH₄ gas,-   (2) of decreasing the production of H₂S, a toxic product, involved    in initiating and/or developing digestive pathologies,-   (3) of promoting the production of metabolites which are healthy for    the host.

In particular, a strain of the Ruminococcus, Clostridium orStreptococcus genus, preferably Ruminococcus hydrogenotrophicus, isused.

Thus, the present invention relates to the use of at least onenonpathogenic, hydrogenotrophic, acetogenic bacterial strain, forpreparing a composition for preventing and/or treating gastrointestinaldisorders by reducing the formation of potentially toxic gases, and/orfor modulating the microbial balance of the digestive ecosystem in amammal. Such food or pharmaceutical compositions are also objects of thepresent invention.

Said reduction and/or said modulation is (are) carried out by decreasingthe amount of gaseous hydrogen (H₂) and/or of gaseous carbon dioxide(CO₂) produced during digestive fermentations.

Said reduction and/or modulation may also be carried out by increasingthe activity of the acetogenic flora in the colon to the detriment ofthe methanogenic and/or sulfate-reducing flora.

The gastrointestinal disorders that the use of a composition containingat least one hydrogenotrophic, acetogenic bacterial strain proposes toreduce are included in the group of functional gastrointestinaldisorders, and in particular excessive flatulence, meteorism, bloatingand abdominal pain, which are major criteria characterizing irritablebowel syndrome. The composition containing at least onehydrogenotrophic, acetogenic bacterial strain can also be used in thecase of ulcerative colitis, of inflammatory bowel diseases or of Crohn'sdisease, in order to reduce the volume of gases in the colon, a factorwhich worsens the symptoms of these pathologies.

In a preferred embodiment of the invention, said composition is a foodcomposition which can be used in the production of new foods or foodingredients as defined in EC Regulation No. 258/97, and in particular inthe manufacture of functional foods. A food may be considered to befunctional if it is demonstrated satisfactorily that it exerts abeneficial effect on one or more target functions in the organism,beyond the usual nutritional effects, improving the state of health andof well-being and/or reducing the risk of a disease (Diplock et al.Scientific concepts of functional foods in Europe: consensus document,British Journal of Nutrition, 1999, 81, S1-S27).

Said composition may in particular constitute a probiotic packaged, forexample, in the form of a capsule or a gelatin capsule.

It is thus possible to use a food composition according to the inventionwhich contains a nonpathogenic, hydrogenotrophic, acetogenic strain andwhich gives the user a feeling of well-being by reducing digestivediscomfort.

In another preferred embodiment of the invention, said composition is apharmaceutical composition, also combined with a pharmaceuticallyacceptable carrier, which may comprise excipients. It is preferablyadministered orally or directly in situ, in particular by coloscopy, orrectally via suppositories.

In another embodiment of the invention, the pharmaceutical compositionalso comprises at least one other agent active against at least one ofthe pathologies targeted.

The pharmaceutical or food composition according to the invention may beadministered orally, in the form of gelatin capsules, of capsules, oftablets, of powders, of granules or of oral solutions or suspensions.The at least one bacterial strain can be mixed with conventionalexcipients, such as gelatin, starch, lactose, magnesium stearate, talc,gum arabic and the like. It may also be advantageous to use lessconventional excipients, which make it possible to increase the abilityof the at least one bacterial strain used to be active in the colon. Forexample, cellobiose, maltose, mannose, salicine, trehalose, amygdalin,arabinose, melobiose, rhamnose and/or xylose may be added. This list isnot exhaustive and the substrates are chosen and adapted as a functionof the strain considered. These substrates may promote heterotrophicand/or mixotrophic growth of the at least one acetogenic strain presentin the composition.

Thus, the composition preferably comprises at least one additive whichpromotes the activity of the at least one strain in the digestiveenvironment.

In a particular embodiment of the invention, the at least onenonpathogenic, hydrogenotrophic, acetogenic bacterial strain present inthe pharmaceutical and/or food composition is administered in a formwhich allows it to be active in the colon. In particular, it isnecessary for the at least one nonpathogenic, hydrogenotrophic,acetogenic bacterial strain to be alive, or viable, in the digestivetract, and in particular the colon. After production of the at least onenonpathogenic, hydrogenotrophic, acetogenic bacterial strain, anddepending on the methods of production, it is also possible to maintainthis strain under anaerobic packaging conditions in order to enable itto remain viable.

In a preferred embodiment, the at least one nonpathogenic,hydrogenotrophic, acetogenic bacterial strain is packaged in ananaerobic environment, i.e. it is packaged in an oxygen-free atmosphere.

In another preferred embodiment of the invention, the at least onenonpathogenic, hydrogenotrophic, acetogenic bacterial strain present inthe composition is an autologous strain of said mammal, i.e. it can beisolated from the digestive system, in particular from the feces, ofother mammals belonging to the same genus.

Preferably, and in particular when the mammal is a human, the at leastone nonpathogenic, hydrogenotrophic, acetogenic bacterial strain belongsto the Ruminococcus genus, even more preferably to the speciesRuminococcus hydrogenotrophicus. Other hydrogenotrophic, acetogenicbacteria, in particular bacteria of the Streptococcus or Clostridiumgenus, in particular Clostridium coccoides, can also be used.

The invention also relates to a method for specifically monitoring thenonpathogenic, hydrogenotrophic, acetogenic bacterial strain in thedigestive tract of a mammal, after it has been used as defined above,comprising the following steps:

-   -   a. a nucleotide sequence (probe) specific for the nonpathogenic,        hydrogenotrophic, acetogenic strain the detection of which is        desired is defined;    -   b. said strain is detected and/or quantified by hybridization of        the probe with total nucleic acid extracted from the fecal        flora, or with the fecal bacteria attached to a slide.

In order to carry out such a monitoring method, diagnostic kits, whichare also objects of the present invention, can be developed. Such kitscontain in particular a “standard” in order to be able to evaluate theamount of bacteria in the feces.

Those skilled in the art are capable of defining a specific sequencewhich does not hybridize with the DNA of other bacteria. Similarly,those skilled in the art will choose hybridization on a membrane or insitu hybridization depending on the means available to them, and on thedesired precision.

Preferably, the nucleic acid detected is the total bacterial DNA, butcan also be a mixture of DNA or of RNA, or the bacterial RNA alone.

The presence of the at least one nonpathogenic, hydrogenotrophic,acetogenic bacterial strain can also be studied using other methods.Detection of the nucleic acids (DNA and/or RNA) of the at least onenonpathogenic, hydrogenotrophic, acetogenic bacterial strain in thefeces of the mammal, in particular by PCR or RT-PCR or by hybridizationwith specific probes (Southern or Northern) will make it possible todetect the presence of said strain and, optionally, the expression ofcertain genes. An advantageous specific sequence can be chosen in thesequence of the gene encoding the 16S rRNA, in particular a region whichis not present on the other species of the flora in the colon.

The invention also covers a method for producing a nonpathogenic,hydrogenotrophic, acetogenic bacterial strain, for its use as definedabove, characterized in that it comprises the following steps:

-   -   a. the strain is grown on a suitable medium, under conditions of        strict anaerobiosis, in the presence of a carbon-based substrate        and/or of H₂/CO₂ as energy source;    -   b. the bacterial cells are recovered;    -   c. the bacterial cells are packaged according to the        pharmaceutical form chosen.

The strain will preferably be grown in a modified AC21 medium (describedin example 1), at 37° C., in a fermenter. The carbon-based substrate maybe glucose.

A preferred method for recovering the bacterial cells is centrifugation,for example between 10 000 g and 15 000 g, advantageously 12 000 g, for15 to 20 minutes. Those skilled in the art are capable of optimizingthese parameters.

The bacteria may advantageously be washed between steps b and c, inparticular in an anaerobic phosphate buffer, by resuspension of thecells, agitation, and a further centrifugation step.

The bacterial pellet, which may or may not be washed, is packaged as afunction of the pharmaceutical form chosen. An advantageous method islyophilization.

The following examples make it possible to illustrate the invention butshould not, however, be considered to be limiting.

DESCRIPTION OF THE FIGURES

FIG. 1: Influence of a 14-day treatment with Ruminococcushydrogenotrophicus on the amounts of hydrogen excreted by rats withhuman flora in a normal nutritional situation.

FIG. 2: Influence of a 14-day treatment with Ruminococcushydrogenotrophicus on the amounts of hydrogen excreted by rats withhuman flora after administration of lactulose.

EXAMPLES Example 1 Isolation of the Microorganisms

Human fecal samples from healthy non-methane-excreting volunteers areused. Individuals are considered to be non-methane-excreting when theirlevel of expired methane does not exceed by more than 1 ppm that ofambient air, which is 1.8 ppm (Bond et al. (1970) Gastroenterology 58 p1035), and when the number of methanogens contained in their fecalextracts is less than 10⁷/g of fecal extract (Bernalier et al. (1996)Arch Microbiol. 166 p 176). The level of methane is determined using achromatograph equipped with a flame ionization detector. The freshlytaken fecal samples are kept at 4° C. under strict anaerobiosis for amaximum of 10 hours.

The enriching, isolating and culturing of the microorganisms are carriedout on a semi-synthetic medium, modified AC-21 medium (Breznak et al.(1988) Arch. Microbiol. 150 p 282) under strict anaerobiosis (Hungate(1969) in: Norris J R and Gibbons D W (eds) Methods in microbiology Vol.3B, New York: Academic Press p 117). The composition per liter of thesemi-synthetic medium is as follows:

KH₂PO₄ 0.2 g NH₄Cl 0.25 g KCl 0.5 g CaCl₂ • 2H₂O 0.15 g MgCl₂ • 6H₂O 0.6g Na₂SO₄ 0.1 q Yeast extract 0.5 g Tryptone 2 g Trace element solution 1ml Tungstate-selenium solution 0.1 ml Vitamin solution 5 ml Resazurinsolution (1%, w/v) 1 ml NaHCO₃ (1 M) 30 ml Cysteine/sulfide reductivesolution 20 ml (1.25%/1.25%, w/v)

The trace element solution is prepared according to Widdel et al. (1983,Arch. Microbiol., 134, p 286), and the vitamin solution is preparedaccording to Greening and Leedle (1989, Arch. Microbiol., 151, p 399).The tungstate-selenium solution has the following composition: 0.1 mMNa₂WO₄ and 0.1 mM Na₂SeO₃, in 20 mM NaOH.

The semi-synthetic medium is solidified by adding a gelling agent (agarat 2%). After inoculation, the gas of the culture medium is replacedwith H₂/CO₂ or N₂/CO₂ depending on the test envisioned.

The dilution medium is a purely inorganic anaerobic medium (Doré et al.(1995) FEMS Microbiol. Lett. 130 p 7). A stock solution is prepared(diluted to one tenth, w/v) from a fecal sample. A series of ten-folddilutions is prepared from the stock suspension. The dilutions thusprepared are inoculated into the semi-synthetic liquid medium containingH₂/CO₂ (60:40, v/v, 202 kPa) as the only energy source (Doré et al.(1995) FEMS Microbiol. Lett. 130 p 7; Bernalier et al. (1996) FEMSMicrobiol. Ecol. 19 p 193; Bernalier et al. (1996) Curr. Microbiol. 33 p94). After incubation at 37° C. for 20 days, the enrichments areobtained from the highest dilution tubes exhibiting the highestbacterial growth, gas consumption and stoichiometric production ofacetate (Bernalier et al. (1996) Curr. Microbiol. 33 p 94). The decreasein gas pressure in the cultures is determined by directly measuringpartial pressure with a manometer of the Capsuhelic type (Dwyer,Instruments, Michigan City, Mich., USA). After three transfers of theenriched cultures, bacterial colonies are isolated using the method ofroll-tubes (Hungate (1969) in: Norris J R and Gibbons D W (eds) Methodsin microbiology Vol. 3B, New York: Academic Press p 117) containing ahomologous agar medium and H₂/CO₂ as energy source. After incubation for20 days at 39° C., the colonies are transferred into liquid media.Purification of the cultures is obtained after 3 to 5 successivetransfers in roll-tubes, at the end of which the purity of the culturesis determined by phase-contrast microscopy, after Gram staining.

The total DNA of the isolated hydrogenotrophic, acetogenic strains isextracted by the method of Lawson et al. (1989, FEMS Microbiol. Lett. 65p 41). The gene encoding the 16S ribosomal RNA is then amplified by PCRusing the universal primers ARI and pH. The PCR products are purifiedand then sequenced using a “Dye-Dideoxy Terminator cycle Sequence” kitand an Applied Biosystem model 373A automatic sequencer. The search for16S rRNA sequence homology between the isolated acetogenic strains andthe other species is carried out using the FASTA program, the sequencedatabases being those of EMBL and of RDP, and using the suggested basicparameters. The sequence alignments are verified manually.

Using this method, it was possible to isolate a bacterium of theRuminococcus genus, identified as being the species Ruminococcushydrogenotrophicus. This bacterium was deposited with the DeutscheSammlung von Mikroorganismen [German Microorganism Collection](Mascheroder Weg 1b, 38124 Braunschweig, Germany) under the number DSM10507, and also under the number DSM 14294, on May 10, 2001 (Treaty ofBudapest).

Example 2 Study of the General Characteristics

-   1—The membrane type of the bacteria is determined by Gram staining    (conventional method) and by the KOH test according to Buck (1982    App. Environ. Microbiol. 44 p 992).-   2—The catalase activity is measured by mixing 1 ml of bacterial    suspension with a few drops of H₂O₂ (30%). The production of gas    bubbles being released more or less strongly indicates the presence    of a catalase.-   3—The cytochrome oxidase activity is studied by placing a bacterial    colony on a disk of filter paper saturated with dimethyl    p-phenylenediamine. A red/purple coloration occurring immediately on    the disk indicates that the test is positive.-   4—The morphological characteristics of the cultures are studied by    phase-contrast microscopy and by electron microscopy after negative    staining with 2% uranyl acetate. The cells are pre-fixed in 2%    glutaraldehyde (15 h at 4° C.) and then fixed with 2% OsO₄ (4° C.,    for a maximum of 15 h). The cells are then embedded in EPPON-812 and    the blocks are very thinly sectioned. These sections are contrasted    with uranyl acetate, soaked in acetate salt and observed with a    transmission electron microscope (Philips 400).-   5—The respiratory type of the bacteria is studied by determining    growth in the presence or absence of O₂.-   6—The effect of variations in pH on bacterial growth is studied by    modifying the CO₂/NaHCO₃ ratio of the semi-synthetic medium    (Costilow (1981) American Society for Microbiology, Washington D.C.    p 66). The bacterial growth is measured (DO600) after 24 or 48 h of    incubation at 37° C. Investigation of the optimal growth temperature    is carried out on semi-synthetic medium containing glucose and the    growth is observed at temperatures ranging from 20 to 45° C. Each    experiment is carried out in triplicate. Bacterial growth is    monitored using a Spectronic 20D spectrophotometer (Bioblock    Scientific, Illkirch, France).

It was found that Ruminococcus hydrogenotrophicus DSM 10507 is anunsporulated, strictly anaerobic Gram-positive coccobacillus. Thenegative staining reveals the absence of flagellae. The bacterial cellsare individual or in pairs. The strain does not have catalase orcytochrome oxidase. The optimal growth temperature and pH are,respectively, 35-37° C. and 6.6. The colonies are translucent, white toslightly brown in color with regular edges, circular and between 1 and 2mm in diameter.

Example 3 Growth Test, Determination of the Acetogenic Activities

The ability of various bacterial strains to metabolize H₂/CO₂ and toform acetate is studied. The strains of Ruminococcus hydrogenotrophicusDSM 10507, and also those taxonomically close, Ruminococcus productusDSM 3507, Ruminococcus productus DSM 2950, Ruminococcus hansenii DSM20583 and Clostridium coccoides DSM 935 (DSM numbers corresponding tothe numbers of the organisms deposited with the Deutsche Sammlung vonMikroorganismen), are incubated on the modified AC-21 medium in thepresence of H₂/CO₂ (60:40, v/v, 202 kPa) as energy source. Controlcultures are incubated under N₂/CO₂ (60:40, v/v, 150 kPa). Threecultures (1 control and 2 tests) are prepared for each bacterial strain.The autotrophic growth is determined by incubation for 96 h in thepresence of H₂/CO₂ (60:40, v/v, 202 kPa). Acetate production is measuredusing an enzymatic test (Boehringer Mannheim, Meylan, France), afterincubation for 6 days at 37° C.

H₂/CO₂ consumption is measured

by determination of the gaseous volume consumed

by chromatographic (CPG) analysis of the composition of the gaseousphase.

The heterotrophic growth of the acetogenic strains (OD₆₀₀) is studied byincubating the bacteria for 20 h with glucose (2 g/l) or fructose (2g/l) as the only source of energy.

The glucose fermentation is studied by incubating the cells at 37° C.for 20 h on the semi-synthetic medium containing 2 g/l of glucose and anatmosphere composed of 100% of CO₂. At the end of incubation, thevolatile fatty acids of the supernatant are assayed by chromatography,after conversion to tertiary derivatives of butyldimethyl (Richardson etal. (1989) Lett. Appl. Microbiol. 9 p 5).

It is observed that the doubling time for R. hydrogenotrophicus at 37°C. on modified AC-21 medium in the presence of H₂/CO₂ (60:40, v/v, 202kPa) as substrate is 26.4 h. The doubling times for R.hydrogenotrophicus at 37° C. with glucose or fructose as energy sourceare approximately 2 or 3 h.

It is observed that the bacterial strain studied is acetogenic: itexhibits autotrophic growth in the presence of H₂/CO₂ and producesacetate as major metabolite (Table I). Among the taxonomically closespecies, acetogenic activity (i.e. consumption of H₂/CO₂ and productionof acetate) is found in C. coccoides and much more weakly in R. hanseniiand R. productus.

R. hydrogenotrophicus DM 10507 consumes approximately 120 mM of H₂ after96 hours of culturing at 37° C. (1.25 mM of H₂ consumed per hour). Totalacetate production is then equal to 30 mM (stoichiometry: 4H₂ consumedper acetate formed).

TABLE I Fermentation characteristics of the strains cultured in thepresence of H₂/CO₂ or glucose as the only energy source. R. hydro-genotro- C. R. R. R. phicus coccoides productus productus hansenii DSMDSM DSM DSM DSM Properties 10507 935 3507 2950 20853 Use of ++ + − + +/−H₂/CO₂ H₂/CO₂ FP Acetate ++ + − + +/− Propionate − − − − − Butyrate − −− − − Lactate − − − − − Succinate − − − − − Ethanol − − − − − Glucose FPAcetate ++ + ++ ++ ++ Propionate − − − − − Butyrate − − − − − Lactate +− − − + Succinate − ++ − − + Ethanol + − + + − Symbols: FP: fermentationproducts; ++, major metabolite; +, nonmajor metabolite; +/−, metaboliteproduced in small amount; −, metabolite not produced.

Example 4 Estimation of the Hydrogenotrophic, Acetogenic Capacity of R.hydrogenotrophicus by Measurement of the Incorporation of ¹³CO₂ intoAcetate (NMR Method)

The incorporation of ¹³CO₂ into acetate is measured by NMR using cellsuspensions of R. hydrogenotrophicus incubated in the presence of H₂.The bacteria are cultured in a 1 l flask containing 250 ml of AC21medium as described in example 1, and H₂/CO₂ as the only energy source.After growth at 37° C., the bacterial cells are recovered bycentrifugation (12 000 g for 20 minutes) and resuspended in a phosphatebuffer containing 20 mM of NaH¹³CO₃ (Leclerc et al (1997), Anaerobe, 3,p 307). The suspensions are incubated for 20 hours at 37° C., under anatmosphere composed of 100% of N₂ (control) or of H₂/N₂ (80/20, v/v), at101 atm. At the end of incubation, the suspensions are again centrifugedat 12 000 g for 20 minutes and the supernatants are recovered. Totalacetate production is measured by an enzymatic method(Boehringer-Mannheim kit). The ¹³C-labeled metabolites are analyzed byNMR (Bernalier et al, (1996), FEMS Microbiol. Ecol., 19, p 193).

When the bacterium is incubated in the presence of H₂, the onlymetabolite detected by ¹³C NMR is acetate. The ¹³C-acetate is labeled inan equivalent manner on its methyl and carboxyl groups. Thedouble-labeled acetate represents 72% of the total labeled acetate. Thisconfirms that the synthesis of acetate from H₂ and CO₂ by R.hydrogenotrophicus occurs via the reductive pathway for acetogenesis.

Example 5 Determination of the Nutritional Capacities of the AcetogenicBacteria

The metabolism of other organic substrates by the acetogenic bacteria isevaluated using a modified AC-21 medium containing 5 or 10 mM ofsubstrate, in an atmosphere composed of 100% CO₂. The test is consideredto be positive when the bacterium maintains its growth after threesuccessive transfers on a medium containing the same substrate, and ifthe OD₆₀₀ of the culture is at least equal to double that observed witha basal semi-synthetic medium (free of organic substrate), afterincubation for 24 h at 37° C.

It is observed that many organic substrates allow heterotrophic growthof the bacteria considered (Table II).

TABLE II Growth of R. hydrogenotrophicus and of the taxonomically closespecies in the presence of various organic substrates R. hydro- genotro-C. R. R. R. phicus coccoides productus productus hansenii DSM DSM DSMDSM DSM Substrate 10507 935 3507 2950 20853 Starch − − + + − Amygdalin− + + + +/− Arabinose − + + + − Cellobiose + + + + − Fructose + + + + −Galactose + + + + + Inulin − − − − + Lactose + + + + + Maltose+/− + + + + Mannitol − + + + − Mannose + + + + − Melibiose +/− + + + +Raffinose + + + + + Rhamnose − + − − − Salicine + + + + − Sorbitol− + + + − Sucrose − + + + − Trehalose + + + + + Xylose − + + + −Symbols: −, no visible growth; +/−, weak growth; +, good growth.

Furthermore, the growth of various hydrogenotrophic, acetogenic strainsisolated from human stools, in the presence of various aromaticcompounds such as vanillate, caffeate or syringate, is estimated bymeasuring the optical density at 600 nm using a Spectronic 20Dspectrophotometer (the effect of adding H₂ to the gaseous phase of thesecultures is also studied). The amount of degraded substrate isdetermined by HPLC, as is the nature of the metabolites formed.

The ability to metabolize the various aromatic substrates tested dependson the hydrogenotrophic, acetogenic strain considered. The Clostridiumspecies are capable of growing and of degrading 20% to 30% of thecaffeate and of the syringate, this degradation reaching 100% when H₂ isadded to the culture. One of the strains of Clostridium degradesvanillate only in the presence of H₂ in the gaseous phase. This straindemethylates the vanillate to protocatechuate in a first step, and then,using H₂, decarboxylates this compound to catechol. The R.hydrogenotrophicus strain exhibits only weak activity with respect tovanillate, this metabolism not appearing to be influenced by thepresence of H₂.

The ability of R. hydrogenotrophicus to use 2 organic substrates notable to be digested by the host's enzymes, fructo-oligosaccharides (FOS)and lactulose, is also determined according to the protocol described inthis example [measurement of bacterial growth (optical density at 600nm) observed in the presence of 2 g/l of each of the indigestiblesubstrates, and maintenance of this growth after 3 successive transferson the same substrate]. R. hydrogenotrophicus proves to be incapable ofmetabolizing these 2 substrates.

Example 6 Mixotrophic Nature of the Hydrogenotrophic, Acetogenic StrainRuminococcus Hydrogenotrophicus

In order to determine whether R. hydrogenotrophicus is capable ofgrowing by mixotrophy (simultaneous use of an organic substrate and ofan inorganic substrate), the strain is cultured in the presence of twoenergy sources, one organic, glucose, and the other inorganic, H₂/CO₂.The culture medium (modified AC21 medium as described in example 1)contains 1.4 mM of glucose and the gaseous phase is replaced, afterseeding, with a mixture of H₂/CO₂ (60/40 at 156 kPa). The cultures areinoculated with 0.3 ml of a preculture of R. hydrogenotrophicus obtainedeither with glucose or with H₂/CO₂ as the only energy source. Theconsumption of gas by the strain is monitored during the incubation at37° C., using pressure sensors attached to the culture stoppers (Leclercet al. (1997), Anaerobe 3 p 307). Bacterial growth is estimated bymeasuring the optical density of the cultures at 600 nm using aSpectronic 20D spectrophotometer. Culture supernatant samples are takenevery 2 hours in order to estimate glucose consumption (assay ofremaining glucose using Boehringer enzymatic method) and fermentativemetabolite production by chromatographic assaying (as described inexample 3).

R. hydrogenotrophicus proves to be capable of co-using the twosubstrates tested. A single exponential growth phase for the bacteriumis observed in the presence of the two substrates. During thisexponential phase, simultaneous consumption of glucose and of H₂/CO₂ isobserved. This reflects the mixotrophic nature of R. hydrogenotrophicuswith regard to these 2 substrates (Leclerc and Bernalier, submitted forpublication). At the end of the exponential phase, the glucose has beencompletely consumed by the bacterium, which bacterium then maintains itsmetabolism in the stationary growth phase by using the remaining H₂/CO₂.

The ability of R. hydrogenotrophicus to cometabolize a sweetener,fructose, and H₂/CO₂, is also studied. The strain is cultured on theAC21 medium, as described in example 1, in the presence of these twoenergy sources. Various concentrations of fructose are tested (2, 1, 0.5and 0.25 g/l), the gaseous phase still being composed of H₂/CO₂ (60/40,v/v, 202 kPa). The experimental protocol then used is the same as thatdescribed above.

R. hydrogenotrophicus proves to be capable of cometabolizing fructoseand H₂/CO₂, the bacterial growth being characterized by a singleexponential growth phase in the presence of the two substrates. Thismixotrophic growth is observed whatever the concentration of fructosepresent in the culture medium and whatever the origin of the bacterialinoculum (preculture prepared on fructose or H₂/CO₂ or fructose+H₂CO₂).In the stationary growth phase, the bacterium metabolizes only H₂/CO₂,all the fructose having been consumed.

Example 7 Effect of Lyophilizing the Ruminococcus hydrogenotrophicusCultures on Expression of the Hydrogenotrophic Activity

In order to determine whether conserving R. hydrogenotrophicus inlyophilized form may impair its ability to use H₂/CO₂, variousconditions for culturing, for conserving the lyophilizates, and forresuspending the bacterium are tested.

R. hydrogenotrophicus cultures are prepared in 1 l flasks containing 250ml of AC21 medium as described in example 1, with fructose (2 g/l),H₂/CO₂ (60/40, v/v, 100 kPa) or both substrates as energy source. Thesecultures are incubated at 37° C. for 24 h, 48 h or 72 h, depending onthe substrate(s) used. Bacterial pellets are then obtained bycentrifugation (15 300×g, 30 min, 4° C.) of the cultures and are takenup in 10 ml of anaerobic buffer. The suspensions thus obtained arealiquoted by 2 ml and then centrifuged for 5 min at 14 000×g. Thebacterial pellets are then frozen at −80° C. before being lyophilizedovernight. The lyophilizates are stored at 4° C. under an aerobic oranaerobic (100% of CO₂) atmosphere. After storage for 15 to 30 days, theR. hydrogenotrophicus lyophilizates are taken up in 5 ml of anaerobicdilution buffer. These bacterial suspensions are then seeded into anAC21 medium containing H₂/CO₂ (60/40, v/v, 200 kPa) as the only energysource either directly or after enrichment for 48 h at 37° C., in acomplex culture medium containing various carbon sources. Afterincubation for 72 h at 37° C. in the presence of H₂/CO₂, the consumptionof H₂ and the amount of acetate produced are determined for eachculture.

Lyophilization of R. hydrogenotrophicus does not appear to affect itshydrogenotrophic potential. The reason for this is that, whatever thesubstrate used to preculture the strain (fructose, H₂/CO₂ or bothsubstrates simultaneously), the hydrogenotrophic activity of R.hydrogenotrophicus is restored when the lyophilizates are placed inculture. Similarly, the aerobic or anaerobic method of storing thelyophilizate has little influence on the expression of thehydrogenotrophic potential of the bacterium. However, maximumhydrogenotrophic activity is observed when R. hydrogenotrophicus isprecultured in the presence of fructose and when the lyophilizate isstored under an anaerobic atmosphere. Similarly, prior culturing of thelyophilized bacteria on a medium rich in organic substratessubstantially increases the expression of their hydrogenotrophicpotential, whatever the substrate used to preculture the strain andwhatever the method of storage of the lyophilizate. This effect isprobably explained by the increase in bacterial density engendered bythe enrichment of the lyophilized cultures on complex medium.

All the results obtained demonstrate that the R. hydrogenotrophicusstrain can be cultured in the presence of organic substrate and thenstored at 4° C. in lyophilized form in an aerobic or anaerobicatmosphere, without this substantially affecting the subsequentexpression of its hydrogenotrophic potential.

Example 8 Interspecies Transfer of H₂, in vitro, Between H₂-ProducingFibrolytic Bacteria and R. hydrogenotrophicus

The ability of R. hydrogenotrophicus to use the H ₂ produced byfibrolytic bacterial species is studied in vitro, in coculturescombining the acetogenic strain with a cellulolytic bacterium. The twocellulolytic species studied were isolated in the laboratory from humanstools. The cocultures are prepared on a semi-synthetic medium,developed in the laboratory, containing a small cellulose strip ofWhatman No. 1 filter paper as the only carbon and energy source. Eachone of these cellulolytic species is seeded in a proportion of 0.5 ml ofinoculum per culture tube. After incubation for 48 h at 37° C., 0.5 mlof R. hydrogenotrophicus inoculum is added to each one of these culturesof cellulolytic bacteria. Control monocultures of each cellulolyticspecies are prepared in parallel.

Kinetics are produced by incubating the cultures at 37° C. for 12 days.After incubation, the amount of H₂ in the gaseous phase is analyzed bychromatography, the amount of cellulose degraded is estimated bymeasuring the remaining solids and the final products of fermentation ofthe cellulose are determined by gas phase chromatography and/or byenzymatic pathways (Roche kit).

The addition of R. hydrogenotrophicus to one or other of theH₂-producing cellulolytic species results in a large decrease in thisgas in the cocultures, whereas it accumulates in the gaseous phase ofthe monocultures.

R. hydrogenotrophicus is therefore capable of effectively re-using theH₂ produced in vitro by a fibrolytic species during fermentation ofcellulose. This transfer of H₂ between R. hydrogenotrophicus and thecellulolytic species causes only slight or no modification of thecellulolytic activity of the fibrolytic species studied. On the otherhand, the cellulose is mainly fermented to acetate in the cocultures,whereas it is rather of the mixed-acid type in the monocultures(production of acetate, of succinate or of ethanol and of lactate).

Example 9 Ability of the Hydrogenotrophic, Acetogenic StrainRuminococcus hydrogenotrophicus to Reduce the Volume of Gases in theColon In Vivo, in Rats with Human Flora

In order to determine whether R. hydrogenotrophicus is capable ofreducing the volume of gases in the colon in the presence of a complexdigestive flora, R. hydrogenotrophicus is administered orally to ratswith human flora and the change in the volume of gas in the colon ismonitored by measuring the hydrogen excreted via the respiratory andrectal pathways.

The effect of R. hydrogenotrophicus is studied in a normal nutritionalsituation and after administration of a fermentable substrate(lactulose) causing an abrupt increase in the volume of gas in thecolon.

The animals used are male Fischer 344 rats, 3 months old at thebeginning of the experiment. They are born free of microorganisms, andare inoculated per os with 1 ml of a centesimal suspension of freshhuman fecal matter from a nonmethanogenic adult donor having aWestern-type diet. The inoculation takes place 2 weeks before thebeginning of the experiment. The rats are housed in isolators andreceive a semi-synthetic “human-type” feed (proteins and fats of animaland plant origins; raw and cooked, simple and complex carbohydrates),sterilized by γ-irradiation at 45 kGy. Food and drinking water aredistributed ad libitum.

Normal Nutritional Situation

The experiment is carried out with 16 rats with human flora, dividedinto 2 groups of 8, a control group and a treated group. The rats in thetreated group receive, each morning for 28 days, by gastric intubation,a dose of 10⁸ to 10⁹ bacteria in the form of 1 ml of an R.hydrogenotrophicus culture cultured for 18 h on AC21 medium (asdescribed in example 1) containing glucose (2 g/l). The control groupreceives, under the same conditions, 1 ml of sterile AC21 mediumcontaining glucose.

After 1, 14 and 28 days of treatment, the rats in the 2 groups areplaced in individual respiratory chambers for 12 h, making it possibleto measure the hydrogen excreted via the respiratory and rectal pathways(see description of the respiratory chambers below). Air samples aretaken in duplicate from the respiratory chambers between 0 h and 12 h.The hydrogen concentration is immediately determined by gas phasechromatography. The results of the control and treated groups arecompared using an ANOVA for repeated measurements.

Whatever its duration of treatment, the administration of R.hydrogenotrophicus significantly decreases the maximum amount ofhydrogen excreted (−50% to −80%) and the excretion rate (−60% to −80%)compared to the control group (P<0.01). The treatment proves to be aseffective after 1, 14 or 28 days (FIG. 1).

The hydrogenotrophic, acetogenic flora is counted (Doré et al, 1995) inthe feces of the rats of the two groups, at various times during theexperiment. In the control group which does not receive R.hydrogenotrophicus, the level of the acetogenic population is stableover time and comparable to that of the donor, i.e. approximately log6.8 acetogens per g of feces.

The treatment with R. hydrogenotrophicus leads to an increase in thelevel of acetogenic flora, which then reaches log 7.5/g of feces from 24h of treatment. This level is maintained until the end of theexperiment. The increase in acetogenic flora in the treated ratstherefore coincides with the decrease in the amounts of H₂ excreted bythese animals.

At the end of the experiment, the rats are sacrificed by euthanasia andautopsied. The administration of R. hydrogenotrophicus has nosignificant effect on either the bodyweight or the weight of the liver,the kidneys and the cecum, or on the pH of the cecum (P>0.05).Similarly, the macroscopic appearance of the liver and of the kidneys ofthe rats treated with R. hydrogenotrophicus is normal and identical tothat of the control rats.

b) After Administration of Lactulose

This experiment is carried out according to the same protocol as above.Lactulose (4-O-β-D-galactopyranosyl-D-fructose), the fermentation ofwhich in the colon leads to an abrupt increase in the volume of gas, isadministered by gastric intubation, in a proportion of 500 mg per rat,just before they are placed in respiratory chambers. The effect of R.hydrogenotrophicus on this increase is examined after 1 and 14 days oftreatment with the acetogenic strain.

Under these conditions of excessive production of gases in the colon,the administration of R. hydrogenotrophicus significantly decreases themaximum amount of hydrogen excreted (−40% to −50%) and the excretionrate (−40% to −50%) compared to the control group (P<0.05). Thetreatment proves to be as effective after 1 or 14 days (FIG. 2).

In the 2 experiments, the rats treated with R. hydrogenotrophicus showedno behavioral abnormality and the appearance and consistency of thefeces was identical to those of the control rats.

The respiratory chambers used are sealed autonomous chambers made oftransparent rigid polyvinyl, with a volume of 30 liters, making itpossible to house a cage containing a small animal. They are equippedwith a double door for sealed transfer, making it possible to connectthem to the experimental isolators.

In order to preserve the bacterial status of the animals, they aresterilized before being connected. After transfer of the animal from theisolator to the respiratory chamber, the latter is detached from theisolator and connected to a closed circuit in which the air is pushed,using a peristaltic pump, through antibacterial filters and systems forremoving the CO₂ (absorber containing a solution of potassium hydroxideat 40%) and the water vapor (absorber containing silicagel crystals).The O₂ content in the air is measured with a sensor and kept constant at21% using an amperometric electrode, a control unit and a solenoidvalve.

This device allows accumulation of the gases specific for fermentationsin the colon (hydrogen and, where appropriate, methane), excreted viathe respiratory and rectal pathways.

1. A method of treating gastrointestinal disorders related to theproduction of hydrogen in the gastrointestinal tract in a mammal, themethod comprising administering to a mammal in need thereof acomposition comprising Ruminococcus hydrogenotrophicus wherein saidmethod does not remove any existing enteric microbial flora prior toadministering said composition orally or rectally and wherein saidcomposition modulates the balance of the microbial flora in the colonand reduces the amount of gaseous hydrogen produced by the existingflora.
 2. The method of claim 1, wherein Ruminococcus hydrogenotrophicusremains active in the colon.
 3. The method of claim 1, wherein thecomposition also comprises a pharmaceutically acceptable carrier.
 4. Themethod of claim 1, wherein said mammal is human, canine or feline. 5.The method of claim 1, wherein said gastrointestinal disorder isselected from the group consisting of irritable bowel syndrome,excessive flatulence, meteorism, bloating and abdominal pain.